Synthetic HPV6/11 hybrid L1 DNA encoding human papillomavirus type 11 L1 protein

ABSTRACT

The present invention is directed to a synthetic DNA molecule encoding purified human papillomavirus type 11 L1 protein and derivatives thereof.

CROSS-RELATED TO OTHER APPLICATIONS

This application is a 371 of PCT/US96/04117, filed Mar. 26, 1997 and acontinuation of U.S. Ser. No. 08/413,572 filed Mar. 30, 1995, abandoned,and a continuation of U.S. Ser. No. 08/413,571 filed Mar. 30, 1995,abandoned.

FIELD OF THE INVENTION

The present invention is directed to a synthetic DNA molecule encodingpurified human papillomavirus type 11 L1 protein and derivativesthereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of the construction of the HPV6/11 hybrid L1 gene,using synthetic oligonucleotides.

FIGS. 2A-2D show the nucleotide sequence of the HPV6/11 hybrid (SEQ IDNO:38), published HPV6a (SEQ ID NO:36) and published HPV11 L1 (SEQ IDNO:37) genes.

FIG. 3 shows the bidirectional yeast expression vector pGAL1-10 used toexpress papillomavirus L1 capsid proteins.

FIG. 4 is a Northern analysis of HPV11 L1 mRNA from yeast.

FIG. 5 shows expression of HPV11 L1 protein in yeast by Western analysis(immunoblot).

FIG. 6 shows ELISA reactivities of HPV11 L1 VLPs expressed fromwild-type (wt) HPV11 compared to HPV6/11 hybrid DNA.

FIG. 7 is an electron micrograph of HPV11 L1 VLPs expressed in yeast.

FIGS. 8A-8B shows the nucleotide sequence of the HPV6/11 hybrid gene(SEQ ID NO:34).

BACKGROUND OF THE INVENTION

Papillomavirus (PV) infections occur in a variety of animals, includinghumans, sheep, dogs, cats, rabbits, monkeys, snakes and cows.Papillomaviruses infect epithelial cells, generally inducing benignepithelial or fibroepithelial tumors at the site of infection. PV arespecies specific infective agents; a human papillomavirus cannot infecta nonhuman animal.

Papillomaviruses may be classified into distinct groups based on thehost that they infect. Human papillomaviruses (HPV) are furtherclassified into more than 70 types based on DNA sequence homology. PVtypes appear to be type-specific immunogens in that a neutralizingimmunity to infection by one type of papillomavirus does not conferimmunity against another type of papillomavirus.

In humans, different HPV types cause distinct diseases. HPV types 1, 2,3, 4, 7, 10 and 26-29 cause benign warts in both normal andimmunocompromised individuals. HPV types 5, 8, 9, 12, 14, 15, 17, 19-25,36 and 46-50 cause flat lesions in immunocompromised individuals. HPVtypes 6, 11, 34, 39, 41-44 and 51-55 cause nonmalignant condylomata ofthe genital or respiratory mucosa. HPV types 16, 18, 31, 33, 35, 45, and58 cause epithelial dysplasia of the genital mucosa and are associatedwith the majority of in situ and invasive carcinomas of the cervix,vagina, vulva and anal canal.

Papillomaviruses are small (50-60 nm), nonenveloped, icosahedral DNAviruses that encode for up to eight early and two late genes. The openreading frames (ORFs) of the virus genomes are designated E1 to E8 andL1 and L2, where "E" denotes early and "L" denotes late. L1 and L2 codefor virus capsid proteins. The early (E) genes are associated withfunctions such as viral replication, transcriptional regulation andcellular transformation.

The L1 protein is the major capsid protein and has a molecular weight of55-60 kDa. L2 protein is a minor capsid protein which has a predictedmolecular weight of 55-60 kDa and an apparent molecular weight of 75-100kDa as determined by polyacrylamide gel electrophoresis. Immunologicaldata suggest that most of the L2 protein is internal to the L1 proteinwithin the viral capsomere. The L1 ORF is highly conserved amongdifferent papillomaviruses. The L2 proteins are less conserved amongdifferent papillomaviruses.

The L1 and L2 genes have been identified as good targets forimmunoprophylaxis. Some of the early genes have also been demonstratedto be potential targets of vaccine development. Studies in thecottontail rabbit papillomavirus (CRPV) and bovine papillomavirus (BPV)systems have shown that immunizations with recombinant L1 and/or L2proteins (produced in bacteria or by using vaccinia vectors) protectedanimals from viral infection. Expression of papillomavirus L1 genes inbaculovirus expression systems or using vaccinia vectors resulted in theassembly of virus-like particles (VLP) which have been used to inducehigh-titer virus-neutralizing antibody responses that correlate withprotection from viral challenge. Furthermore, the L1 and L2 genes havebeen used to generate vaccines for the prevention and treatment ofpapillomavirus infections in animals.

The development and commercialization of prophylactic and therapeuticvaccines for PV infection and disease containing L1 protein, L1+L2proteins, or modified L1 or L1+L2 proteins has been hindered by the lackof large quantities of purified virus and purified protein. Because PVis not readily cultivated in vitro, it is difficult to produce therequired amounts of L1 and L2 protein by in vitro propagation of PV. Theresultant supply problems make it difficult to characterize PV and PVproteins. Accordingly, it would be useful to develop a readily renewablesource of crude PV proteins, especially PV L1 and L2 proteins ormodified L1 and L2 proteins. It would also be useful to develop methodsof purifying large quantities of the crude papillomavirus proteins tolevels of purity suitable for immunological studies and vaccinedevelopment. It would also be useful to produce large quantities ofpapillomavirus proteins having the immunity-conferring properties of thenative proteins, such as the conformation of the native protein. Inaddition, it would be useful to develop methods of analyzing the PVproteins and methods of determining the relative purity of the proteinsas well as compositions containing the proteins. Such highly purifiedproteins would also be useful in the preparation of a variety ofreagents useful in the study of PV infection; such reagents include butare not limited to polyclonal antibodies, monoclonal antibodies, andanalytical standards.

HPV6 and 11 are causative agents for ˜90% of benign genital warts andare only rarely associated with malignancies (Gissmann et al., 1983,PNAS 80, 560-563). HPV6a is considered to be the most abundant HPV6subtype in condyloma accuminata (Brown, D. B., et al., J. Clin.Microbiol. 31:1667-1673). Office visits for genital warts (condylomaaccuminatum or planum) have been on the rise in recent years. It isestimated that ˜10% of the general population (ages 15-49) havegenital-tract HPV infections (Koutsky et al. 1988, Epidemiol. Rev. 10,122-163). While the majority of condylomata is associated with HPV6, inthe case of laryngeal papillomatosis, HPV11 is the dominant type. HPV11replication in the epithelial cells of the respiratory tract stimulatesthe proliferation of these cells which can lead to isolated lesions ofminor clinical relevance or to multiple spreading lesions and recurringdisease. Recurrent respiratory papillomatosis, a disease which moreoften afflicts the juvenile population, can be a life-threateningdisease by causing obstructions in the respiratory tract. Recently, ananimal model which allows the replication of infectious HPV11, has beendeveloped (Kreider et al. 1985, Nature 317, 639-640; Kreider et al.1987, J. Virol. 61, 590-593). The model enabled the identification ofconformational neutralizing epitopes on native virions andbaculoviru-sexpressed VLPs using monoclonal antibodies (Christensen etal 1990, J. Virol. 64, 5678-5681; Christensen and Kreider 1991, VirusRes. 21, 169-179; Christensen and Kreider 1993, Virus Res. 28, 195-202;Christensen et al. 1994, 75, 2271-2276).

Virus-like particles containing HPV11 L1 protein have been expressed inboth insect and mammalian cell systems. Expression of VLPs in yeastcells offers the advantages of being cost-effective and easily adaptedto large-scale growth in fermenters. However, the HPV11 L1 protein isexpressed at low levels in yeast cells. This was observed to be a resultof truncation of the HPV11 L1 mRNA. In contrast, the HPV6 L1 gene istranscribed as full-length mRNA and is expressed to high levels. Bymodifying the HPV6 L1 DNA to encode the HPV11 L1 protein, it is possibleto facilitate the transcription of full-length mRNA resulting inincreased HPV11 L1 protein expression. The present invention provides anHPV6/11 hybrid L1 gene sequence as well as a method for the constructionof the HPV6/11 hybrid L1 gene using synthetic oligonucleotides. Thehybrid gene was designed using the HPV6a L1 sequence (Hofiann, K. J., etal., 1995, Virology, accepted for publication) but contains the minimalnumber of base changes necessary to encode the HPV11 L1 protein. Unlikethe wild-type HPV11 L1 gene, the HPV6/11 hybrid gene does not containyeast-recognized internal transcription termination signals; as a resultfull-length HPV6/11 mRNA is produced and expression of HPV11 L1 proteinis increased.

The present invention is directed to highly purified PV L1 protein. Theinvention also comprises methods by which recombinant papillomavirusproteins having the immunity-conferring properties of the nativepapillomavirus proteins are produced and purified. The present inventionis directed to the production of prophylactic and therapeutic vaccinesfor papillomavirus infection. Electron microscopy and binding toconformational antibodies demonstrate that the recombinant proteins ofthe present invention are capable of forming virus-like particles.

SUMMARY OF THE INVENTION

The present invention is directed to a synthetic DNA molecule encodingpurified human papillomavirus type 11 L1 protein and derivativesthereof.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a synthetic DNA molecule encodingpurified human papillomavirus type 11 L1 protein and derivativesthereof. Various embodiments of the invention include but are notlimited to recombinant HPV DNA molecules, RNA complementary to therecombinant HPV DNA molecules, proteins encoded by the recombinant DNAmolecules, antibodies to the recombinant DNA molecules and relatedproteins, compositions comprising the DNA, RNA, proteins orantibodies,methods of using the DNA, RNA, proteins or antibodies as well asderivatives thereof. Such derivatives include but are not limited topeptides and proteins encoded by the DNA, antibodies to the DNA orantibodies to the proteins encoded by the DNA, vaccines comprising theDNA or vaccines comprising proteins encoded by the DNA, immunologicalcompositions comprising the DNA or the proteins encoded by the DNA, kitscontaining the DNA or RNA derived from the DNA or proteins encoded bythe DNA.

HPV6 and 11 are causative agents for ˜90% of benign genital warts andare only rarely associated with malignancies (Gissmann et al., 1983,PNAS 80, 560-563). Office visits for genital warts (condylomaaccuminatum or planum) have been on the rise in the last years and it isestimated that ˜10% of the general population (ages 15-49) havegenital-tract HPV infections (Koutsky et al. 1988, Epidemiol. Rev. 10,122-163). While the majority of condylomata is associated with HPV6, inthe case of laryngeal papillomatosis, HPV11 is the dominant type. HPV11replication in the epithelial cells of the respiratory tract stimulatesthe proliferation of these cells which can lead to isolated lesions ofminor clinical relevance or to multiple spreading lesions and recurringdisease. Recurrent respiratory papillomatosis, a disease which moreoften afflicts the juvenile population, can be a life-threateningdisease by causing obstructions in the respiratory tract. Recently, ananimal model which allows the replication of infectious HPV11, has beendeveloped (Kreider et al. 1985, Nature 317, 639-640; Kreider et al.1987, J. Virol. 61, 590-593). The model enabled the identification ofconformational neutralizing epitopes on native virions andbaculovirus-expressed VLPs using monoclonal antibodies (Christensen etal. 1990, J. Virol 64, 5678-5681; Christensen and Kreider 1991,Virus-Res. 21, 169-179; Christensen and Kreider 1993, Virus Res. 28,195-202; Christensen et al. 1994, 75, 2271-2276).

The development and commercialization of prophylactic and therapeuticvaccines for PV infection and disease containing L1 protein, L1+L2proteins, or modified L1 or L1+L2 proteins has been hindered by the lackof large quantities of purified virus and purified protein. Because PVis not readily cultivated in vitro, it is difficult to produce therequired amounts of L1 and L2 protein by in vitro propagation of PV. Thedifficulties associated with in vitro cultivation of PV also result indifficulties in chemical, immunological and biological characterizationof PV and PV proteins. Accordingly, it would be useful to develop areadily renewable source of crude PV proteins, especially PV L1 and L2proteins or modified L1 and L2 proteins. It would also be useful todevelop methods of purifying large quantities of the crudepapillomavirus proteins to levels of purity suitable for immunologicalstudies and vaccine development. It would also be useful to producelarge quantities of papillomavirus proteins having theimmunity-conferring properties of the native proteins, such as theconformation of the native protein. In addition, it would be useful todevelop methods of analyzing the PV proteins and methods of determiningthe relative purity of the proteins as well as compositions containingthe proteins. Such highly purified proteins would also be useful in thepreparation of a variety of reagents useful in the study of PVinfection; such reagents include but are not limited to polyclonalantibodies, monoclonal antibodies, and analytical standards.

Virus-like particles containing HPV11 L1 protein have been expressed inboth insect and mammalian cell systems. Expression of VLPs in yeastcells offers the advantages of being cost-effective and easily adaptedto large-scale growth in fermenters. However, the HPV11 L1 protein isexpressed at low levels in yeast cells. This was observed to be a resultof truncation of the HPV11 L1 mRNA. In contrast, the HPV6 L1 gene istranscribed as full-length mRNA and is expressed to high levels. Bymodifying the HPV6 L1 DNA to encode the HPV11 L1 protein, it is possibleto facilitate the transcription of full-length mRNA resulting inincreased HPV11 L1 protein expression. The present invention provides anHPV6/11 hybrid L1 gene as well as a method for the construction of theHPV6/11 hybrid L1 gene using synthetic oligonucleotides. The hybrid genewas designed using the HPV6a L1 sequence but contains the minimal numberof base changes necessary to encode the HPV11 L1 protein. Unlike thewild-type HPV11 L1 gene, the HPV6/11 hybrid gene does not containyeast-recognized internal transcription termination signals, resultingin higher levels of mRNA and consequently increased HPV11 L1 proteinexpression.

Pharmaceutically useful compositions comprising the DNA or proteinsencoded by the DNA may be formulated according to known methods such asby the admixture of a pharmaceutically acceptable carrier. Examples ofsuch carriers and methods of formulation may be found in Remington'sPharmaceutical Sciences. To form a pharmaceutically acceptablecomposition suitable for effective administration, such compositionswill contain an effective amount of the protein or VLP. Suchcompositions may contain proteins or VLP derived from more than one typeof HPV.

Therapeutic or diagnostic compositions of the invention are administeredto an individual in amounts sufficient to treat or diagnose PVinfections. The effective amount may vary according to a variety offactors such as the individual's condition, weight, sex and age. Otherfactors include the mode of administration. Generally, the compositionswill be administered in dosages ranging from about 1 mcg to about 1 mg.

The pharmaceutical compositions may be provided to the individual by avariety of routes such as subcutaneous, topical, oral, mucosal,intravenous and intramuscular.

The vaccines of the invention comprise DNA, RNA or proteins encoded bythe DNA that contain the antigenic determinants necessary to induce theformation of neutralizing antibodies in the host. Such vaccines are alsosafe enough to be administered without danger of clinical infection; donot have toxic side effects; can be administered by an effective route;are stable; and are compatible with vaccine carriers.

The vaccines may be administered by a variety of routes, such as orally,parenterally, subcutaneously, mucosally, intravenously orintramuscularly. The dosage administered may vary with the condition,sex, weight, and age of the individual; the route of administration; andthe type PV of the vaccine. The vaccine may be used in dosage forms suchas capsules, suspensions, elixirs, or liquid solutions. The vaccine maybe formulated with an immunologically acceptable carrier.

The vaccines are administered in therapeutically effective amounts, thatis, in amounts sufficient to generate a immunologically protectiveresponse. The therapeutically effective amount may vary according to thetype of PV. The vaccine may be administered in single or multiple doses.

The purified proteins of the present invention may be used in theformulation of immunogenic compositions. Such compositions, whenintroduced into a suitable host, are capable of inducing an immuneresponse in the host.

The purified proteins of the invention or derivatives thereof may beused to generate antibodies. The term "antibody" as used herein includesboth polyclonal and monoclonal antibodies, as well as fragments thereof,such as, Fv, Fab and F(ab)2 fragments that are capable of bindingantigen or hapten.

The proteins and protein derivatives of the present invention may beused to serotype HPV infection and HPV screening. The purified proteins,VLP and antibodies lend themselves to the formulation of kits suitablefor the detection and serotyping of HPV. Such a kit would comprise acompartmentalized carrier suitable to hold in close confinement at leastone container. The carrier may further comprise reagents such as L1 orL2 proteins or VLPs derived from recombinant HPV6/11 or otherrecombinant HPV type DNA molecules or antibodies directed against theseproteins. The carrier may also contain means for detection such aslabeled antigen or enzyme substrates or the like.

The purified proteins are also useful as immunological standards,molecular weight markers and molecular size markers.

It is known that there is a substantial amount of redundancy in thevarious codons which code for specific amino acids. Therefore, thisinvention is also directed to those DNA sequences which containalternative codons which code for the eventual translation of theidentical amino acid. For purposes of this specification, a sequencebearing one or more replaced codons will be defined as a degeneratevariation. Also included within the scope of this invention aremutations either in the DNA sequence or the translated protein which donot substantially alter the ultimate physical properties of theexpressed protein. For example, substitution of valine for leucine,arginine for lysine, or asparagine for glutamine may not cause a changein functionality of the polypeptide.

It is known that DNA sequences coding for a peptide may be altered so asto code for a peptide having properties that are different than those ofthe naturally-occurring peptide. Methods of altering the DNA sequencesinclude, but are not limited to site directed mutagenesis.

As used herein, a "functional derivative" of the HPV6/11 hybrid gene isa compound that possesses a biological activity (either functional orstructural) that is substantially similar to the biological activity ofHPV6/11. The term "functional derivatives" is intended to include the"fragments," "variants," "degenerate variants," "analogs" and"homologues" or to "chemical derivatives" of HPV6/11.

The term "analog" refers to a molecule substantially similar in functionto either the entire HPV6/11 molecule or to a fragment thereof.

The cloned HPV6/11 DNA or fragments thereof obtained through the methodsdescribed herein may be recombinantly expressed by molecular cloninginto an expression vector containing a suitable promoter and otherappropriate transcription regulatory elements, and transferred intoprokaryotic or eukaryotic host cells to produce recombinant HPV11.Techniques for such manipulations are fully described in Sambrook, J.,et al., supra, and are known in the art.

Expression vectors are defined herein as DNA sequences that are requiredfor the transcription of cloned copies of genes and the translation oftheir mRNAs in an appropriate host. Such vectors can be used to expresseukaryotic genes in a variety of hosts such as bacteria, bluegreenalgae, plant cells, insect cells, fungal cells and animal cells.Specifically designed vectors allow the shuttling of DNA between hostssuch as bacteria-yeast or bacteria-animal cells or bacteria-fungal cellsor bacteria-invertebrate cells. An appropriately constructed expressionvector should contain: an origin of replication for autonomousreplication in host cells, selectable markers, a limited number ofuseful restriction enzyme sites, a potential for high copy number, andactive promoters. A promoter is defined as a DNA sequence that directsRNA polymerase to bind to DNA and initiate RNA synthesis. A strongpromoter is one which causes mRNAs to be initiated at high frequency.Expression vectors may include, but are not limited to, cloning vectors,modified cloning vectors, specifically designed plasmids or viruses.

A variety of mammalian expression vectors may be used to express HPV6/11DNA or fragments thereof in mammalian cells. Commercially availablemammalian expression vectors which may be suitable for recombinantHPV6/11 DNA expression, include but are not limited to, pcDNA3(Invitrogen), pMC1neo (Stratagene), pXT1 (Stratagene), pSG5(Stratagene), EBO-pSV2-neo (ATCC 37593) pBPV-1(8-2) (ATCC 37110),pdBPV-MMTneo(342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460), and λZD35 (ATCC37565).

A variety of bacterial expression vectors may be used to express HPV6/11DNA or fragments thereof in bacterial cells. Commercially availablebacterial expression vectors which may be suitable for recombinantHPV6/11 DNA expression include, but are not limited to pET11a (Novagen),lambda gt11 (Invitrogen), pcDNAII (Invitrogen), pKK223-3 (Pharmacia).

A variety of fungal cell expression vectors may be used to expressHPV6/11 or fragments thereof in fungal cells. Commercially availablefungal cell expression vectors which may be suitable for recombinantHPV6/11 DNA expression include but are not limited to pYES2(Invitrogen), Pichia expression vector (Invitrogen) and Hansenulaexpression (Rhein Biotech, Dusseldorf, Germany).

A variety of insect cell expression vectors may be used to expressHPV6/11 DNA or fragments thereof in insect cells. Commercially availableinsect cell expression vectors which may be suitable for recombinantexpression of HPV6/11 DNA include but are not limited to pBlue Bac III(Invitrogen).

An expression vector containing HPV6/11 DNA or fragments thereof may beused for expression of HPV11 proteins or fragments of HPV11 proteins ina cell, tissue, organ, or animal. Animal, as used herein, includeshumans. Host cells may be prokaryotic or eukaryotic, including but notlimited to bacteria such as E. Coli, fungal cells such as yeast,mammalian cells including but not limited to cell lines of human,bovine, porcine, monkey and rodent origin, and insect cells includingbut not limited to Drosophila and silkworm derived cell lines. Celllines derived from mammalian species which may be suitable and which arecommercially available, include but are not limited to, L cells L-M(TK⁻)(ATCC CCL 1.3), L cells L-M (ATCC CCL 1.2), 293 (ATCC CRL 1573), Raji(ATCC CCL 86), CV-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCCCRL 1651), CHO-K1 (ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL1658), HeLa (ATCC CCL 2), C127I (ATCC CRL 1616), BS-C-1 (ATCC CCL 26)and MRC-5 (ATCC CCL 171).

The expression vector may be introduced into host cells via any one of anumber of techniques including but not limited to transformation,transfection, lipofection, protoplast fusion, and electroporation. Theexpression vector-containing cells are clonally propagated andindividually analyzed to determine whether they produce HPV11 protein.Identification of HPV11 expressing host cell clones may be done byseveral means, including but not limited to immunological reactivitywith anti-HPV11 antibodies.

Expression of HPV DNA fragments may also be performed using in vitroproduced synthetic mRNA or native mRNA. Synthetic mRNA or mRNA isolatedfrom cells expressing HPV6/11 hybrid DNA can be efficiently translatedin various cell-free systems, including but not limited to wheat germextracts and reticulocyte extracts, as well as efficiently translated incell based systems, including but not limited to microinjection intofrog oocytes, with microinjection into frog oocytes being preferred.

Following expression of HPV11 protein in a host cell, HPV11 protein maybe recovered to provide HPV11 protein in purified form. Several HPV11purification procedures are available and suitable for use. As describedherein, recombinant HPV11 protein may be purified from cell lysates andextracts by various combinations of, or individual application of saltfractionation, ion exchange chromatography, size exclusionchromatography, hydroxylapatite adsorption chromatography andhydrophobic interaction chromatography.

In addition, recombinant HPV11 protein may be separated from othercellular proteins by use of an immunoaffinity column made withmonoclonal or polyclonal antibodies specific for full length nascentHPV11, or polypeptide fragments of HPV11. Monoclonal and polyclonalantibodies may be prepared according to a variety of methods known inthe art. Monoclonal or monospecific antibody as used herein is definedas a single antibody species or multiple antibody species withhomogenous binding characteristics for HPV11. Homogenous binding as usedherein refers to the ability of the antibody species to bind to aspecific antigen or epitope.

It is apparent to those skilled in the art that the methods forproducing monospecific antibodies may be utilized to produce antibodiesspecific for HPV polypeptide fragments, or full-length nascent HPVpolypeptides. Specifically, it is apparent to those skilled in the artthat monospecific antibodies may be generated which are specific for thefully functional HPV proteins or fragments thereof.

The present invention is also directed to methods for screening forcompounds which modulate the expression of DNA or RNA encoding HPV aswell as the function(s) of HPV11 protein in vivo. Compounds whichmodulate these activities may be DNA, RNA, peptides, proteins, ornon-proteinaceous organic molecules. Compounds may modulate byincreasing or attenuating the expression of DNA or RNA encoding HPV11,or the function of HPV11 protein. Compounds that modulate the expressionof DNA or RNA encoding HPV11 or the function of HPV11 protein may bedetected by a variety of assays. The assay may be a simple "yes/no"assay to determine whether there is a change in expression or function.The assay may be made quantitative by comparing the expression orfunction of a test sample with the levels of expression or function in astandard sample.

Kits containing HPV6/11 hybrid DNA, fragments of HPV6/11 hybrid DNA,antibodies to HPV6/11 DNA or HPV11 protein, HPV6/11 hybrid RNA or HPV11protein may be prepared. Such kits are used to detect DNA whichhybridizes to HPV6/11 DNA or to detect the presence of HPV11 protein orpeptide fragments in a sample. Such characterization is useful for avariety of purposes including but not limited to forensic analyses andepidemiological studies.

Nucleotide sequences that are complementary to the HPV6/11 DNA sequencemay be synthesized for antisense therapy. These antisense molecules maybe DNA, stable derivatives of DNA such as phosphorothioates ormethylphosphonates, RNA, stable derivatives of RNA such as2'-O-alkylRNA, or other HPV6/11 antisense oligonucleotide mimetics.HPV6/11 antisense molecules may be introduced into cells bymicroinjection, liposome encapsulation or by expression from vectorsharboring the antisense sequence. HPV6/11 antisense therapy may beparticularly useful for the treatment of diseases where it is beneficialto reduce HPV11 activity.

The term "chemical derivative" describes a molecule that containsadditional chemical moieties which are not normally a part of the basemolecule. Such moieties may improve the solubility, half-life,absorption, etc. of the base molecule. Alternatively the moieties mayattenuate undesirable side effects of the base molecule or decrease thetoxicity of the base molecule. Examples of such moieties are describedin a variety of texts, such as Remington's Pharmaceutical Sciences.

Compounds identified according to the methods disclosed herein may beused alone at appropriate dosages defined by routine testing in order toobtain optimal inhibition of the HPV11 or its activity while minimizingany potential toxicity. In addition, co-administration or sequentialadministration of other agents may be desirable.

Advantageously, compounds of the present invention may be administeredin a single dose, or the total dosage may be administered in severaldivided doses. Furthermore, compounds for the present invention may beadministered via a variety of routes including but not limited tointranasally, transdermally, by suppository, orally, and the like.

For combination treatment with more than one active agent, where theactive agents are in separate dosage formulations, the active agents canbe administered concurrently, or they each can be administered atseparately staggered times.

The dosage regimen utilizing the compounds of the present invention isselected in accordance with a variety of factors including type,species, age, weight, sex and medical condition of the patient; theseverity of the condition to be treated; the route of administration;the renal and hepatic function of the patient; and the particularcompound thereof employed. A physician of ordinary skill can readilydetermine and prescribe the effective amount of the drug required toprevent, counter or arrest the progress of the condition. Optimalprecision in achieving concentrations of drug within the range thatyields efficacy without toxicity requires a regimen based on thekinetics of the drug's availability to target sites. This involves aconsideration of the distribution, equilibrium, and elimination of adrug.

In the methods of the present invention, the compounds herein describedin detail can form the active ingredient, and may be administered inadmixture with suitable pharmaceutical diluents, excipients or carriers(collectively referred to herein as "carrier" materials) suitablyselected with respect to the intended form of administration, that is,oral tablets, capsules, elixirs, syrup, suppositories, gels and thelike, and consistent with conventional pharmaceutical practices.

For instance, for oral administration in the form of a tablet orcapsule, the active drug component can be combined with an oral,non-toxic pharmaceutically acceptable inert carrier such as ethanol,glycerol, water and the like. Moreover, when desired or necessary,suitable binders, lubricants, disintegrating agents and coloring agentscan also be incorporated into the mixture. Suitable binders includewithout limitation, starch, gelatin, natural sugars such as glucose orbeta-lactose, corn sweeteners, natural and synthetic gums such asacacia, tragacanth, sodium alginate, carboxymethylcellulose,polyethylene glycol, waxes and the like. Lubricants used in these dosageforms include, without limitation, sodium oleate, sodium stearate,magnesium stearate, sodium benzoate, sodium acetate, sodium chloride andthe like. Disintegrators include, without limitation, starch, methylcellulose, agar, bentonite, xanthan gum and the like.

For liquid forms the active drug component can be combined in suitablyflavored suspending or dispersing agents such as the synthetic andnatural gums, for example, tragacanth, acacia, methyl-cellulose and thelike. Other dispersing agents which may be employed include glycerin andthe like. For parenteral administration, sterile suspensions andsolutions are desired. Isotonic preparations which generally containsuitable preservatives are employed when intravenous administration isdesired.

Topical preparations containing the active drug component can be admixedwith a variety of carrier materials well known in the art, such as,e.g., alcohols, aloe vera gel, allantoin, glycerine, vitamin A and Eoils, mineral oil, PPG2 myristyl propionate, and the like, to form,e.g., alcoholic solutions, topical cleansers, cleansing creams, skingels, skin lotions, and shampoos in cream or gel formulations.

The compounds of the present invention can also be administered in theform of liposome delivery systems, such as small unilamellar vesicles,large unilamellar vesicles and multilamellar vesicles. Liposomes can beformed from a variety of phospholipids, such as cholesterol,stearylamine or phosphatidylcholines.

Compounds of the present invention may also be delivered by the use ofmonoclonal antibodies as individual carriers to which the compoundmolecules are coupled. The compounds of the present invention may alsobe coupled with soluble polymers as targetable drug carriers. Suchpolymers can include polyvinyl-pyrrolidone, pyran copolymer,polyhydroxypropylmethacryl-amidephenol,polyhydroxyethylaspartamidephenol, or polyethyl-eneoxidepolylysinesubstituted with palmitoyl residues. Furthermore, the compounds of thepresent invention may be coupled to a class of biodegradable polymersuseful in achieving controlled release of a drug, for example,polylactic acid, polyepsilon caprolactone, polyhydroxy butyric acid,polyorthoesters, polyacetals, polydihydro-pyrans, polycyanoacrylates andcross-linked or amphipathic block copolymers of hydrogels.

The following examples illustrate the present invention without,however, limiting the same thereto.

EXAMPLE 1

Construction of the Synthetic L1 Gene

The 1.5 kbp open reading frame of HPV11 L1 was constructed usingsynthetic DNA oligomers ordered from Midland Reagent Company. Theseoligomers were supplied containing 5' terninal phosphates. A total of 24oligomers were required and are listed below:

    #241-1                  (SEQ ID NO:1)                                         5'GAAGATCTCACAAAACAAAATGTGGCGGCCTAGC                                          GACAGCACAGTATATGTGCCTCCTCCTAACCCTGT                                           ATCCAAAGTTGTTGCCACGGATGCTTATGTTAAAC                                           GCACCAACATATTTTATCATGCCAGCAGTTCTAGA                                           CTTCTTGCAGTGGGTCATCCTTATT3'                                                   #2412                   (SEQ ID NO:2) - 5'ATTCCATAAAAAAGGTTAACAAAACTGTTGTG                            CC                                                    AAAGGTGTCAGGATATCAATACAGAGTATTTAAGG                                           TGGTGTTACCAGATCCTAACAAATTTGCATTGCCT                                           GACTCGTCTCTTTTTGATCCCACAACACAACGTTT                                           GGTATGGGCATGCATGT3'                                                           #241-3                  (SEQ ID NO:3)                                         5'ACATGCATGCACAGGCCTAGAGGTGGGCCGGGGA                                          CAGCCATTAGGTGTGGGTGTAAGTGGACATCCTTT                                           ACTAAATAAATATGATGATGTTGAAAATTCAGGGG                                           GTTACGGTGGTAACCCTGGACAGGATAACAGG3'                                            #241-4                  (SEQ ID NO:4)                                         5'GTTAATGTAGGTATGGATTATAAACAAACACAAT                                          TATGCATGGTTGGATGTGCCCCCCCTTTGGGCGAG                                           CATTGGGGTAAAGGTACACAGTGTAGTAATACATC                                           TGTACAGAATGGTGACTGCCCGC3'                                                     #241-5                  (SEQ ID NO:5)                                         5'CCTTAGAACTTATTACCAGTGTTATACAGGATGG                                          CGATATGGTTGACACAGGCTTTGGTGCTATGAATT                                           TTGCTGATTTGCAGACCAATAAATCAGATGTTCCT                                           CTTGACATATGTGGCACTGTA3'                                                       #241-6                  (SEQ ID NO:6)                                         5'TGTAAATATCCAGATTATTTACAAATGGCTGCAG                                          ACCCATATGGTGATAGATTATTTTTTTATCTACGG                                           AAGGAACAAATGTTTGCCAGACATTTTTTTAACAG                                           GGCTGGTACCCC3'                                                                #241-7                  (SEQ ID NO:7)                                         5'GGGGTACCGTGGGGGAACCTGTGCCTGATGATCT                                          TTTAGTTAAGGGTGGTAACAATCGCTCGTCTGTAG                                           CGAGTAGTATATATGTTCACACCCCAAGCGGCTCT                                           TTGGTGTCCTCTGAGGCACA3'                                                        #241-8                  (SEQ ID NO:8)                                         5'ATTGTTTAATAAGCCATATTGGCTACAAAAAGCC                                          CAGGGACATAACAATGGTATTTGTTGGGGTAATCA                                           TCTGTTTGTTACTGTGGTAGATACCACACGCAGTA                                           CCAACATGA3'                                                                   #241-9                  (SEQ ID NO:9)                                         5'CATTATGTGCATCCGTATCTAAATCTGCCACATA                                          CACCAATTCTGATTATAAAGAGTACATGCGTCATG                                           TGGAAGAGTTTGATTTACAATTTATTTTTCAATTA                                           TGTAGCATT3'                                                                   #241-10                 (SEQ ID NO:10)                                        5'ACATTGTCTGCTGAAGTAATGGCCTATATTCACA                                          CAATGAATCCCTCTGTTCTCGAGGACTGGAACTTT                                           GGGTTATCGCCTCCCCCAAATGGTACACTCGAGCG                                           G3'                                                                           #241-11                 (SEQ ID NO:11)                                        5'CCGCTCGAGGATACCTATAGGTATGTGCAGTCAC                                          AGGCCATTACCTGTCAAAAGCCCACTCCTGAAAAG                                           GAAAAGCAAGATCCCTATAAGGACATGAGTTTTTG                                           GGAGGTTAATTTAAAAGAAAAGTTTTCTAGTGAAT                                           TGGATCAGTTTCCTTT3'                                                            #241-12                 (SEQ ID NO:12)                                        5'GGGACGCAAGTTTTTGTTACAAAGTGGATATAGG                                          GGACGGACCTCTGCTCGTACCGGTATTAAGCGCCC                                           TGCTGTTTCCAAACCCTCTACTGCCCCTAAACGTA                                           AGCGCACCAAAACTAAAAAGTAAGATCTTC3'                                              #241-13                 (SEQ ID NO:13)                                        5'GAAGATCTTACTTTTTAGTTTTGGTGCGCTTACG                                          TTTAGGGGCAGTAGAGGGTTTGGAAACAGCAGGGC                                           GCTTAATACCGGTACGAGCAGAGGTCCGTCCCCTA                                           TATCCACTTTGTAACAAAAACTTGCGTCCCAAAGG                                           AAACTGATCCAATTC3'                                                             #241-14                 (SEQ ID NO:14)                                        5'ACTAGAAAACTTTTCTTTTAAATTAACCTCCCAA                                          AAACTCATGTCCTTATAGGGATCTTGCTTTTCCTT                                           TTCAGGAGTGGGCTTTTGACAGGTAATGGCCTGTG                                           ACTGCACATACCTATAGGTATCCTCGAGCGG3'                                             #241-15                 (SEQ ID NO:15)                                        5'CCGCTCGAGTGTACCATTTGGGGGAGGCGATAAC                                          CCAAAGTTCCAGTCCTCGAGAACAGAGGGATTCAT                                           TGTGTGAATATAGGCCATTACTTCAGCAGACAATG                                           TAATGCTACATAATTGAAAAA3'                                                       #241-16                 (SEQ ID NO:16)                                        5'TAAATTGTAAATCAAACTCTTCCACATGACGCAT                                          GTACTCTTTATAATCAGAATTGGTGTATGTGGCAG                                           ATTTAGATACGGATGCACATAATGTCATGTTGGTA                                           CTGCGTGTG3'                                                                   #241-17                 (SEQ ID NO:17)                                        5'GTATCTACCACAGTAACAAACAGATGATTACCCC                                          AACAAATACCATTGTTATGTCCCTGGGCTTTTTGT                                           AGCCAATATGGCTTATTAAACAATTGTGCCTCAGA                                           GGACACCAA3'                                                                   #241-18                 (SEQ ID NO:18)                                        5'AGAGCCGCTTGGGGTGTGAACATATATACTACTC                                          GCTACAGACGAGCGATTGTTACCACCCTTAACTAA                                           AAGATCATCAGGCACAGGTTCCCCCACGGTACCCC                                           3'                                                                            #241-19                 (SEQ ID NO:19)                                        5'GGGGTACCAGCCCTGTTAAAAAAATGTCTGGCAA                                          ACATTTGTTCCTTCCGTAGATAAAAAAATAATCTA                                           TCACCATATGGGTCTGCAGCCATTTGTAAATAATC                                           TGGATATTTACATACAGTGCCACATATGTCAA3'                                            #241-20                 (SEQ ID NO:20)                                        5'GAGGAACATCTGATTTATTGGTCTGCAAATCAGC                                          AAAATTCATAGCACCAAAGCCTGTGTCAACCATAT                                           CGCCATCCTGTATAACACTGGTAATAAGTTCTAAG                                           GGCGGGCAGTCACCATTCTGT3'                                                       #241-21                 (SEQ ID NO:21)                                        5'ACAGATGTATTACTACACTGTGTACCTTTACCCC                                          AATGCTCGCCCAAAGGGGGGGCACATCCAACCATG                                           CATAATTGTGTTTGTTTATAATCCATACCTACATT                                           AACCCTGTTATCCTGTCCAGGGT3'                                                     #241-22                 (SEQ ID NO:22)                                        5'TACCACCGTAACCCCCTGAATTTTCAACATCATC                                          ATATTTATTTAGTAAAGGATGTCCACTTACACCCA                                           CACCTAATGGCTGTCCCCGGCCCACCTCTAGGCCT                                           GTGCATGCATGT3'                                                                #241-23                 (SEQ ID NO:23)                                        5'ACATGCATGCCCATACCAAACGTTGTGTTGTGGG                                          ATCAAAAAGAGACGAGTCAGGCAATGCAAATTTGT                                           TAGGATCTGGTAACACCACCTTAAATACTCTGTAT                                           TGATATCCTGACACCTTTGGCACAACAGTTTTGTT                                           AACCTTTTTTATGGAATAATAAGGATGACCC3'                                             #241-24                 (SEQ ID NO:24)                                        5'ACTGCAAGAAGTCTAGAACTGCTGGCATGATAAA                                          ATATGTTGGTGCGTTTAACATAAGCATCCGTGGCA                                           ACAACTTTGGATACAGGGTTAGGAGGAGGCACATA                                           TACTGTGCTGTCGCTAGGCCGCCACATTTTGTTTT                                           GTGAGATCTTC3'                                                             

Oligomers forming complementary pairs (#241-1 and #241-24, #241-2 and#241-23, #241-3 and #241-22, #241-4 and #241-21, #241-5 and #241-20,#241-6 and #241-19, #241-7 and #241-18, #241-8 and #241-17, #241-9 and#241-16, #241-10 and #241-15, #241-11 and #241-14, #241-12 and#241-13-FIG. 1) were annealed in separate tubes containing 2.5 mM Tris,pH 7.5, 0.25 mM EDTA. Tubes were heated to 98° C. for 4 min and thenplaced in 200 ml of 98° C. water in a 250 ml beaker to cool slowly. Whenthe water cooled to room temperature, the annealed pairs were added totubes as designated: fragment A (oligomer pairs #241-1 & 24, and -2 &23); fragment B (#241-3 & 22, -4 & 21, -5 & 20, and -6 & 19); fragment C(#241-7 & 18, -8 & 17, -9 & 16 and -10 & 15) and fragment D (#241-11 &14 and -12 & 13). These oligomer pair mixes were heated to 62° C. for 2min and then cooled slowly as before. The contents of each tube wereligated overnight at 23° C. using T4 DNA ligase (Boehringer Mannheim,Inc.) and the reagents supplied by the manufacturer.

After ligation, fragment B required PCR amplification to increase theamount of full-length product. This required ten cycles of 94° C., 1min; 48° C., 1 min; 72° C., 1 min followed by 10 min at 72° C. in anApplied Biosystems theimocycler using Boehringer Mannheim Taq polymeraseand the oligomer primers:

    5'GGAATTCACATGCATGCACAGGCCTAG 3'                                                                      (SEQ ID NO:25)                                        and                                                                           5'GGAATTCGGGGTACCAGCCCTGTTAA 3'                                                                       (SEQ ID NO:26).                                   

The ligated products and the fragment B PCR product were digested withrestriction enzymes (Boehringer Mannheim, Inc.) as follows: fragment Awas digested with Bgl II and Sph I; fragment B, Sph I and Kpn I;fragment C, Kpn I and Xho I; and fragment D, Xho I and Bgl II. Thedigested fragments were separated on low melting point agarose (FMCBioProducts) gels and correctly sized fragments isolated by excision ofthe band and digestion of the agarose using Agarase™ (BoehringerMannheim, Inc.) as recommended by the supplier. The fragments A, B and Dwere recovered by ethanol precipitation and then separately ligated intothe vector pSP72 (Promega, Inc.) that had been similarly digested withrestriction enzymes to match each fragment being ligated.

The Kpn I Xho I digested fragment C was first ligated to the annealedoligomers

    5'TCGAAGACTGGAACTTTGGGTTATCGCCTCCCC                                                                  (SEQ ID NO:27)                                         CAAATGGTACAC3';                                                               and                                                                           5'TCGAGTGTACCATTTGGGGGAGGCGATAACCCA                                                                  (SEQ ID NO:28) - AAGTTCCAGTCT3'.                   

Fragment C was then recleaved with Xho I and the 450 bp Kpn I Xho Ifragment was ligated with the Kpn I, Xho I-digested pSP72 vector. Theligation mixes were used to transform Escherichia coli strain DH5competent cells (Gibco BRL, Gaithersburg, Md.). Transformants werescreened for insert-containing clones by colony hybridization (J.Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition,Cold Spring Harbor Laboratory Press, 1989). Plasmid DNA was isolatedfrom the positive clones using a Wizard miniprep kit (Promega Corp.) andthen sequenced using an Applied Biosystems 373A DNA Sequencer. Clonescontaining the correct DNA sequence for each of the four fragments weredigested as before to release the fragments from the pSP72 vector. TheKpn I, Xho I-digested fragment C was ligated with the Xho I, BglII-digested fragment D and Kpn I, Bgl II-cut pSP72 in a three-wayligation. The ligation products were then used to transform E. coli.Resulting transformants were sequenced and a clone of correct sequenceobtained (designated CD). The 750 bp Bgl II Kpn I insert of CD wasrecleaved from the pSP72 vector and ligated with Bgl II, Sph I digestedfragment A and Sph I, Kpn I-digested fragment B in a threeway ligationas before except Bgl II was added to decrease undesired ligationproducts. The ligation products were separated on agarose gels, the 1.5kbp fragment was isolated, and was designated D361-1.

EXAMPLE 2

Comparison of Sequences

A comparison of the nucleotide sequence for the HPV6/11 hybrid, HPV6aand HPV11 L1 DNA sequences is shown in FIG. 2. There are a total of 55nucleotide substitutions made to the HPV6 backbone sequence to convertit to a HPV11-encoding translation frame. In addition, three base pairinsertions were added at #411-413 bp to encode the additional amino acid(tyrosinel 32) found in HPV11 but not HPV6. Together, these changesallow the type 11-specific, conformation-dependent, neutralizingmonoclonal antibody (Chemicon 8740 MAb) to bind the L1 protein of theHPV6/11 L1 DNA expressed in yeast. This suggests that the protein fromthe HPV6/11 hybrid gene appears to be indistinguishable immunologicallyfrom native HPV11.

Comparison of the HPV6/11 hybrid DNA sequence to the published HPV11 L1sequence shows 194 base pair substitutions. There are a considerablenumber of substitutions relative to the wild type 11 L1 sequence, anycombination of which or all changes in total may be what is responsiblefor the increased type 11 L1 protein expression in yeast.

EXAMPLE 3

DNA Sequencing of the L1 gene

The HPV6/11 L1 gene was sequenced using an Applied Biosystems DNASequencer #373A with dye terminator sequencing reactions (PRIZM™Sequencing Kit) as specified by the manufacturer (ABI, Inc., FosterCity, Calif.).

EXAMPLE 4 Construction of HPV6/11 L1, HPV11 L1 and HPV6 L1 YeastExpression Vectors

The pGAL1-10 yeast expression vector was constructed by isolating a 1.4kbp Sph I fragment from a pUC18/bidirectional GAL promoter plasmid whichcontains the Saccharomyces cerevisiae divergent GAL1-GAL10 promotersfrom the plasmid pBM272 (provided by Mark Johnston, WashingtonUniversity, St. Louis, Mo.). The divergent promoters are flanked on eachside by a copy of the yeast ADH1 transcriptional terminator (Bennetzen,J. L. and Hall, B. D., 1982, J. Biol. Chem. 257: 3018-3025), a BamHIcloning site located between the GAL1 promoter and the first copy of theADH1 transcriptional terminator and a SmaI cloning site located betweenthe GAL10 promoter and the second copy of the ADH1 transcriptionalterminator. A yeast shuttle vector consisting of pBR322, the yeast LEU2dgene (Erhart, E. and Hollenberg, C. P., 1983, J. Bacteriol. 156:625-635) and the yeast 2μ plasmid (gift of Benjamin Hall, University ofWashington, Seattle, Wash.) (Broach, J. R. and Volkert, F. C., 1991,Circular DNA Plasmids of Yeasts, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y.) was digested with Sph I and ligated with the1.4 kbp Sph I divergent GAL promoter fragment resulting in pGAL1-10(FIG. 3).

The HPV6/11 hybrid L1 DNA encoding the HPV11 L1 protein (sample D361-1from Example 1) contains a yeast non-translated leader sequence(Kniskem, P. J. et al., 1986, Gene 46: 135-141) immediately upstream tothe HPV6/11 L1 initiating methionine codon. The pGAL1-10 plasmid waslinearized with BamHI which cuts between the GAL1 promoter and the ADH1transcription terminator and ligated with the 1.5 kbp, HPV6/11 L1 genefragment (sample D361-1). E. coli DH5 (Gibco BRL, Inc.) transformantswere screened and a pGAL1-10 plasmid containing the HPV6/11 L1 gene wasisolated and designated as D362-1.

The wild-type HPV11 (wt-HPV11) DNA was cloned from a condylomaacuminatum lesion (kind gift of Dr. Darron Brown). Total human genomicDNA was extracted and digested with restriction endonucleases. Thefraction containing wt-HPV11 DNA was ligated into an E. coli cloningvector to be used as a template for PCR. The wt-HPV11 L1 gene wasamplified by PCR using Vent polymerase (New England Biolabs, Inc.), 10cycles of amplification (94° C. 1 min, 48° C. 1 min, 72° C. 1 min 45sec), and the following oligonucleotide primers which contain flankingBgl II sites (underlined):

    sense primer:                                                                           5'-CTC AGA TCT CAC AAA                                                                          (SEQ ID NO:29)                                              ACA AAA TGT GGC GGC                                                           CTA GCG ACA GCA CAG-3'                                              antisense 5'-GAG AGA TCT TAC TTT                                                                          (SEQ ID NO:30)                                    primer:   TTG GTT TTG GTA CGT                                                           TTT CG-3'                                                       

The sense primer introduces a yeast non-translated leader sequence(Kniskem, P. J. et al., 1986, Gene 46: 135-141) immediately upstream tothe wt-HPV11 L1 initiating methionine codon (highlighted in bold print).The 1.5 kbp wt-HPV11 L1 PCR product was digested with Bgl II, gelpurified and ligated with the BamHI digested pGAL1-10 plasmid to yieldplasmid, p329-1.

Total genomic DNA was extracted from an HPV6a-positive, condylomaacuminatum lesion (kind gift of Dr. Darron Brown). The HPV6a L1 gene wasamplified by PCR using the biopsy sample DNA as a template, Ventpolymerase (New England Biolabs, Inc.), 35 cycles of amplification (94°C. 1 min, 48° C. 1 min, 72° C. 1 min 45 sec), the sense primer listedabove for PCR of wt-HPV11 L1 and an antisense primer with the sequence,

    (SEQ ID NO:31)                                                                5'-GAG AGA TCT TAC CTT TTA GTT TTG GCG CGC TTA C-3'.                      

The 1.5 kbp HPV6a L1 PCR product was digested with Bgl II, gel purifiedand ligated with the BamHI digested pGAL1-10 plasmid to yield plasmidD128.

EXAMPLE 5

Preparation of Strain 1558

Step a: Preparation of Yeast Strain U9

Saccharomyces cerevisiae strain 2150-2-3 (MATalpha, leu2-04, adel, cir°)was obtained from Dr. Leland Hartwell (University of Washington,Seattle, Wash.). Cells of strain 2150-2-3 were propagated overnight at30° C. in 5 mL of YEHD medium (Carty et al., J. Ind Micro 2 (1987)117-121). The cells were washed 3 times in sterile, distilled water,resuspended in 2 mL of sterile distilled water, and 0.1 mL of cellsuspension was plated onto each of six 5-fluoro-orotic acid (FOA) platesin order to select for ura3 mutants (Cold Spring Harbor LaboratoryManual for Yeast Genetics). The plates were incubated at 30° C. Themedium contained per 250 mL distilled water: 3.5 g, Difco Yeast NitrogenBase without amino acids and ammonium sulfate; 0.5 g 5-Fluoro-oroticacid; 25 mg Uracil; and 10.0 g Dextrose.

The medium was sterilized by filtration through 0.2 μm membranes andthen mixed with 250 mL of 4% Bacto-Agar (Difco) maintained at 50° C., 10mL of a 1.2 mg/mL solution of adenine, and 5 mL of L-leucine solution(180 mg/50 mL). The resulting medium was dispensed at 20 mL per petridish.

After 5 days of incubation, numerous colonies had appeared. Singlecolonies were isolated by restreaking colonies from the initial FOAplates onto fresh FOA plates which were then incubated at 30° C. Anumber of colonies from the second set of FOA plates were tested for thepresence of the ura3 mutation by replica-plating onto both YEHD platesand uracil-minus plates. The desired result was good growth on YEHD andno growth on uracil-minus medium. One isolate (U9) was obtained whichshowed these properties. It was stored as a frozen glycerol stock(strain #325) at -70° C. for later use.

Step b: Preparation of a Vector for disruption of the Yeast MNN9 gene

In order to prepare a vector for disruption of the MNN9 gene, it wasnecessary to first clone the MNN9 gene from S. cerevisiae genomic DNA.This was accomplished by standard Polymerase Chain Reaction (PCR)technology. A 5' sense primer and 3' antisense primer for PCR of thefull-length MNN9 coding sequence were designed based on the publishedsequence for the yeast MNN9 gene (Zymogenetics: EPO Patent ApplicationNo. 88117834.7, Publication No. 0-314-096-A2). The followingoligodeoxynucleotide primers containing flanking HindIII sites(underlined) were used:

    sense primer:                                                                           5'-CTT AAA GCT TAT GTC                                                                          (SEQ ID NO:32)                                              ACT TTC TCT TGT ATC G-3'                                            antisense 5'-TGA TAA GCT TGC TCA                                                                          (SEQ ID NO:33)                                    primer:   ATG GTT CTC TTC CTC-3'                                          

The initiating methionine codon for the MNN9 gene is highlighted in boldprint. The PCR was conducted using genomic DNA from S. cerevisiae strainJRY 188 as template, Taq DNA polymerase (Perkin Elmer) and 25 cycles ofamplification (94° C. 1 min., 37° C. 2 min., 72° C. 3 min.). Theresulting 1.2 kbp PCR fragment was digested with HindIII, gel-purified,and ligated with HindIII-digested, alkaline-phosphatase treated pUC13(Pharmacia). The resulting plasmid was designated p1183.

In order to disrupt the MNN9 gene with the yeast URA3 gene, the plasmidpBR322-URA3 (which contains the 1.1 Kbp HindIII fragment encoding the S.cerevisiae URA3 gene subcloned into the HindIII site of pBR322) wasdigested with HindIII and the 1.1 kbp DNA fragment bearing thefunctional URA3 gene was gel-purified, made bluntended with T4 DNApolymerase, and then ligated with PmlI-digested plasmid p1183 (PmlI cutswithin the MNN9 coding sequence). The resulting plasmid p1199 contains adisruption of the MNN9 gene by the functional URA3 gene.

Step c: Construction of U9-derivative strain 1372 containing disruptionof MNN9 gene

For disruption of the MNN9 gene in strain U9 (#325), 30 μg of plasmidp1199 were digested with HindIII to create a linear mnn9::URA3disruption cassette. Cells of strain 325 were transformed with theHindIII-digested p1199 DNA by the spheroplast method (Hinnen et al.,1978, Proc. Natl. Acad. Sci. USA 75:1929-1933) and transformants wereselected on a synthetic agar medium lacking uracil and containing 1.0Msorbitol. The synthetic medium contained, per liter of distilled water:Agar, 20 g; Yeast nitrogen base w/o amino acids, 6.7 g; Adenine, 0.04 g;L-tyrosine, 0.05 g; Sorbitol, 182 g; Glucose, 20 g; and Leucine MinusSolution #2, 10 ml. Leucine Minus Solution #2 contains per liter ofdistilled water: L-arginine, 2 g; L-histidine, 1 g; L-Leucine, 6 g;L-Isoleucine, 6 g; L-lysine, 4 g; L-methionine, 1 g; L-phenylalanine, 6g; L-threonine, 6 g; L-tryptophan, 4 g.

The plates were incubated at 30° C. for five days at which time numerouscolonies had appeared. Chromosomal DNA preparations were made from 10colonies and then digested with EcoRI plus HindIII. The DNA digests werethen evaluated by Southern blots (J. Sambrook et al., Molecular Cloning:A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press,1989) using the 1.2 kbp HindIII fragment bearing the MNN9 gene (isolatedfrom plasmid p1199) as a probe. An isolate was identified (strain #1372)which showed the expected DNA band shifts on the Southern blot as wellas the extreme dumpiness typically shown by mnn9 mutants.

Step d: Construction of a Vector for Disruption of Yeast HIS3 Gene

In order to construct a disruption cassette in which the S. cerevisiaeHIS3 gene is disrupted by the URA3 gene, the plasmid YEp6 (K. Struhl etal., 1979, Proc. Natl. Acad. Sci., USA 76:1035) was digested with BamHIand the 1.7 kbp BamHI fragment bearing the HIS3 gene was gel-purified,made blunt-ended with T4 DNA polymerase, and ligated with pUC18 whichhad been previously digested with BamHI and treated with T4 DNApolymerase. The resulting plasmid (designated p1501 or pUC18-HIS3) wasdigested with NheI (which cuts in the HIS3 coding sequence), and thevector fragment was gel-purified, made blunt-ended with T4 DNApolymerase, and then treated with calf intestine alkaline phosphatase.The URA3 gene was isolated from the plasmid pBR322-URA3 by digestionwith HindIII and the 1.1 kbp fragment bearing the URA3 gene wasgel-purified, made blunt-ended with T4 DNA polymerase, and ligated withthe above pUC18-HIS3 NheI fragment. The resulting plasmid (designatedpUC18-his3::URA3 or p1505) contains a disruption cassette in which theyeast HIS3 gene is disrupted by the functional URA3 gene.

Step e: Construction of Vector for Disruption of Yeast PRB1 Gene by theHIS3 Gene

Plasmid FP8ΔH bearing the S. cerevisiae PRB1 gene was provided by Dr. E.Jones of Carnegie-Mellon Univ. (C. M. Moehle et al., 1987, Genetics115:255-263). It was digested with HindIII plus Xho I and the 3.2 kbpDNA fragment bearing the PRB1 gene was gel-purified and made blunt-endedby treatment with T4 DNA polymerase. The plasmid pUC18 was digested withBamHI, gel-purified and made blunt-ended by treatment with T4 DNApolymerase. The resulting vector fragment was ligated with the abovePRB1 gene fragment to yield the plasmid pUC18-PRB1. Plasmid YEp6, whichcontains the HIS3 gene, was digested with BamHI. The resulting 1.7 kbpBamHI fragment bearing the functional HIS3 gene was gel-purified andthen made blunt-ended by treatment with T4 DNA polymerase. PlasmidpUC18-PRB1 was digested with EcoRV plus NcoI which cut within the PRB1coding sequence and removes the protease B active site and flankingsequence. The 5.7 kbp EcoRV-NcoI fragment bearing the residual 5' and3'-portions of the PRB1 coding sequence in pUC18 was gel-purified, madeblunt-ended by treatment with T4 DNA polymerase, dephosphorylated withcalf intestine alkaline phosphatase, and ligated with the blunt-endedHIS3 fragment described above. The resulting plasmid (designatedpUC18-prb1::HIS3, stock #1245) contains the functional HIS3 gene inplace of the portion of the PRB1 gene which had been deleted above.

Step f: Construction of a U9-related Yeast Strain containing disruptionsof both the MNN9 and PRB1 Genes

The U9-related strain 1372 which contains a MNN9 gene disruption wasdescribed in Example 5c. Clonal isolates of strain 1372 were passaged onFOA plates (as described in Example 5a) to select ura3 mutants. A numberof ura3 isolates of strain 1372 were obtained and one particular isolate(strain 12930-190-S1-1) was selected for subsequent disruption of theHIS3 gene. The pUC18-his3::URA3 gene disruption vector (p1505) wasdigested with XbaI plus EcoRI to generate a linear his3::URA3 disruptioncassette and used for transformation of strain 12930-190-S1-1 by thelithium acetate method (Methods in Enzymology, 194:290 (1991). Ura⁺transformants were selected on synthetic agar medium lacking uracil,restreaked for clonal isolates on the same medium, and thenreplica-plated onto medium lacking either uracil or histidine to screenfor those isolates that were both Ura⁺ and His⁻. One isolate (strain12930-230-1) was selected for subsequent disruption of the PRB1 gene.The PRB1 gene disruption vector (pUC18-prb1::HIS3, stock #1245) wasdigested with SacI plus XbaI to generate a linear prb1::HIS3 disruptioncassette and used for transformation of strain 12930-230-1 by thelithium acetate method. His⁺ transformants were selected on agar mediumlacking histidine and restreaked on the same medium for clonal isolates.Genomic DNA was prepared from a number of the resulting His⁺ isolates,digested with EcoRI, and then electrophoresed on 0.8% agarose gels.Southern blot analyses were then performed using a radio-labeled 617 bpprobe for the PRB1 gene which had been prepared by PCR using thefollowing oligodeoxynucleotide primers:

    5' TGG TCA TCC CAA ATC TTG AAA 3'                                                                     (SEQ ID NO:34);                                       and                                                                           5' CAC CGT AGT GTT TGG AAG CGA 3'                                                                     (SEQ ID NO:35)                                    

Eleven isolates were obtained which showed the expected hybridization ofthe probe with a 2.44 kbp prb1::HIS3 DNA fragment. This was in contrastto hybridization of the probe with the 1.59 kbp fragment for thewild-type PRB1 gene. One of these isolates containing the desiredprb1::HIS3 disruption was selected for further use and was designatedstrain #1558.

EXAMPLE 6

Expression of HPV11 L1 and HPV6 L1 in Yeast

Plasmids D362-1 (pGAL1-10+HPV6/11 L1), p329-1 (pGAL1-10+wt-HPV11 L1),D128 (pGAL1-10+HPV6 L1) and pGAL1-10 were used to transform S.Cerevisiae strain #1558 (MATa, leu2-04, prb1::HIS3, mnn9::URA3, adel,cir°) by the spheroplast method (Hinnen et al., 1978, Proc. Natl. Acad.Sci. USA 75, 1929-1933). The #1558 yeast strain transformed with plasmidD362-1 was designated as strain #1782. For RNA studies, yeast clonalisolates were grown at 30° C. in YEH complex medium (Carty et al., 1987,J. Ind. Micro. 2, 117-121) containing 0.1M sorbitol and either 2%glucose or galactose for 26 hours. After harvesting the cells, yeast RNAwas extracted using the hot acidic phenol method as described (CurrentProtocols in Molecular Biology, vol. 2, Current Protocols, 1993). Forprotein analysis, the identical isolates were grown at 30° C. in YEHcomplex medium containing 0.1M sorbitol, 2% glucose and 2% galactose for70 hours. After harvesting the cells, the cell pellets were broken withglass beads and cell lysates analyzed for the expression of HPV11 L1 orHPV6 L1 protein by immunoblot analysis.

EXAMPLE 7

Northern Blot Analysis of Yeast Expressed HPV L1 RNAs

Samples containing 10 μg of total RNA were denatured by treatment withglyoxal and DMSO (Current Protocols in Molecular Biology, vol. 1,Current Protocols, 1993) and electrophoresed through aphosphate-buffered, 1.2% agarose gel. The RNA was transferred onto anylon membrane and detected with a ³² P-labeled oligonucleotide that iscomplementary to both the HPV11 and HPV6 L1 DNA sequences.

The Northern blot is shown in FIG. 4. No bands that correspond to theexpected size for full-length HPV L1 RNA were detected in the samplesgrown on glucose medium (lanes 1, 3 and 5). This is expected sinceglucose represses transcription from the yeast GAL1 promoter. Incontrast, samples grown in galactose medium which induces transcriptionfrom the GAL1 promoter, show strong HPV L1 RNA signals. The HPV6 L1 wastranscribed as a full-length RNA species (lane 2) while the majority ofthe wild-type (wt)-HPV11 L1 was transcribed as a truncated form (lane4). This result suggested that a yeast transcription termination signalis located within the wt-HPV11 L1 ORF but is not present in the HPV6 L1sequence. The RNA transcribed from the HPV6/11 hybrid gene appears to befull-length (lane 6). No HPV specific RNA is detected in the pGAL1-10control yeast sample (lane 7).

EXAMPLE 8

Western Analysis of Yeast Expressed HPV L1 Proteins

Samples containing 20 μg of total cellular protein were electrophoresedthrough 10% Tris-Glycine gels (Novex, Inc.) under denaturing conditionsand electroblotted onto nitrocellulose filters. L1 protein wasimmunodetected using rabbit antiserum raised against a trpE-HPV11 L1fusion protein as primary antibody (Brown, D. R. et al., 1994, Virology201:46-54) and horseradish peroxidase (HRP)-linked donkey anti-rabbitIgG (Amersham, Inc.) as secondary antibody. The filters were processedusing the chemiluminescent ECL™ Detection Kit (Amersham, Inc.). A 50-55kDa L1 protein band was detected in all samples except the pGAL1-10negative control (lane 4) (FIG. 5). Furthermore, the amount of HPV11 L1protein expressed by the HPV6/11 hybrid gene (lane 3) appears to be˜10-fold greater than the amount of L1 protein expressed by either thewt-HPV11 gene (lane 2) or the HPV6 L1 gene (lane 1).

EXAMPLE 9

ELISA of Yeast Expressed HPV11 L1 VLPs

An ELISA was used to determine relative amounts of VLPs produced fromyeast clones expressing either wt-HPV11 or the HPV6/11 hybrid. The ELISAwas also used to demonstrate that a conformational epitope giving riseto strongly neutralizing antibody responses was retained on the VLPsderived from the HPV6/11 hybrid DNA. This conformational epitope hasbeen defined by monoclonal antibody H11.B2 (Christensen et al 1990, J.Virol. 64, 5678-5681) which is available from Chemicon International(Temecula, Calif.) as Chemicon Mab8740. Briefly, wells of ELISA plates(Maxisorb, Nunc Inc., Naperville, Ill.) were coated with decreasingamounts of total yeast protein containing the HPV6/11 (hybrid) orwt-HPV11 VLPs in 100 μL PBS. CsCl-purified wt-HPV11 virions (a generousgift of Dr. D. Brown) and control yeast protein were used as controls.The plates were incubated overnight at 4° C. before aspirating andblocking the plates with 250 mcl 10% dried milk (Carnation) in TTBS (50mM Tris, pH 7.6, 150 mM NaCl, 0.1% Tween20) for 2 hrs at roomtermperature. The plates were washed once with PBS/0.1% Tween 20 beforeincubating the wells with 100 mcl of a 1:1000 dilution of the anti-HPV11virion monoclonal antibody Chemicon MAB 8740 in 1% dried milk in TTBSfor 1 hr at room temperature. Plates were washed 3 times with PBS/Tween20 and then incubated with 100 mcl of anti-mouse IgG coupled to alkalinephosphatase (Kierkegard & Perry, Gaithersburg, Md.) at a dilution of1:1000 in 1% milk+TTBS for 1 hr at room temperature. Plates were againwashed 3 times with PBS/Tween 20 before adding 100 mcl of phosphatasesubstrate (p-nitrophenyl phosphate in diethanolamine buffer). Plateswere incubated 30 min at room temperature. The reaction was stopped byaddition of 50 mcl of 3N NaOH. Plates were read at 405 nm in an ELISAplate reader.

The average OD₄₀₅ nm readings of 2 wells corrected against thebackground readings obtained from control yeast proteins were plottedagainst the total yeast protein in the wells and are shown in FIG. 6.The HPV6/11 hybrid yeast clone produced more than 10 times the amount ofnative VLPs compared to the wt clone. In addition, the stronglyneutralizing epitope recognized by Chemicon Mab 8740 is displayed onthese VLPs.

EXAMPLE 10

Electron Microscopic Studies

For EM analysis (Structure Probe, West Chester, Pa.), an aliquot of eachsample (crude clarified lysate or purified VLPs) was placed on 200-meshcarbon-coated copper grids. A drop of 2% phosphotungstic acid (PTA), pH7.0 was placed on the grid for 20seconds. The grids were allowed to airdry prior to transmission EM examination. All microscopy was done usinga JEOL 100CX transmission electron microscope (JEOL USA, Inc.) at anaccelerating voltage of 100 kV. The micrographs generated have a finalmagnification of 100,000×. As shown in FIG. 7, VLPs were observed in the45-55 nm diameter size range in all HPV11 samples but not in yeastcontrol samples.

EXAMPLE 11

Fermentation of HPV6/11 L1 (Strain #1782)

Surface growth of a plate culture of strain 1782 was asepticallytransferred to a leucine-free liquid medium containing (per L): 8.5 gDifco yeast nitrogen base without amino acids and ammonium sulfate; 0.2g adenine; 0.2 g uracil; 10 g succinic acid; 5 g ammonium sulfate; and0.25 g L tyrosine; this medium was adjusted to pH 5.0-5.3 with NaOHprior to sterilization. After growth for 25 hr at 28° C., 250 rpm on arotary shaker, frozen culture vials were prepared by adding sterileglycerol to a final concentration of 17% (w/v) prior to storage at -70°C. (1 mL per cryovial). Inoculum for fermentation of strain 1782 wasdeveloped in the same medium (750 mL per 2-L flask) and was started bytransferring the thawed contents of two frozen culture vials to the 2-Lflasks and incubating at 28° C., 250 rpm on a rotary shaker for 25 hr.Fermentation of strain 1782 used a Chemap 23 L fermenter with a workingvolume of 18 L after inoculation. The production medium used contained(per L): 20 g Difco yeast extract; 10 g Sheffield HySoy peptone; 20 gglucose; 20 g galactose; the medium was adjusted to pH 5.3 prior tosterilization. The entire contents (500 mL) of the 2-L inoculum flaskwas transferred to the fermenter which was incubated at 28° C., 9 L airper min, 500 rpm, 3.5 psi pressure. Agitation was increased as needed tomaintain dissolved oxygen levels of greater than 40% of saturation.Progress of the fermentation was monitored by off line glucosemeasurements (Beckman Glucose 2 Analyzer) and online mass spectrometry(Perkin-Elmer 1200). After 66 hr incubation, a cell density of 9.32 gdry cell weight per L was reached. The contents of two suchfermentations (total 17.5 L broth) were pooled before cell recovery. Theculture was concentrated by hollow fiber filtration (Amicon H5MP01-43cartridge in an Amicon DC-10 filtration system) to ca. 2 L, diafilteredwith 2 L phosphate-buffered saline, and concentrated further (to ca. 1L) before dispensing into 500 mL centrifuge bottles. Cell pellets werecollected by centrifugation at 8,000 rpm (Sorval GS3 rotor) for 20 minat 4° C. After decanting the supernatant, the pellets (total 358 g wetcells) were stored at -70° C. until use.

EXAMPLE 12

Purification of Recombinant HPV Type 11 L1 Capsid Proteins

All steps were performed at 4° C. unless noted.

Cells were stored frozen at -70° C. Frozen cells (wet weight=180 g) werethawed at 20-23° C. and resuspended in 900 mL "Breaking Buffer" (50 mMMOPS, pH 7.2, 500 mM NaCl, 1 mM CaCl₂). The protease inhibitors AEBSFand pepstafin A were added to final concentrations of 1 mM and 1.7 μM,respectively. The cell slurry was broken at a pressure of approximately16,000 psi by 4 passes in a M110-Y Microfluidizer (Microfluidics Corp.,Newton, Mass.). A sufficient volume of 10% Triton X100® detergent(Pierce, Rockford, Ill.) was added to the broken cell slurry to bringthe concentration of TX100 to 0.5%. The slurry was stirred for 20 hours.The Triton X100-treated lysate was centrifuged at 12,000×g for 40 min toremove cellular debris. The supernatant liquid containing L1 protein wasrecovered.

The supernatant liquid was diafiltered against five volumes of 20 mMsodium phosphate, pH 7.2, 0.5M NaCl using a 300K tangential flowmembrane cassette (Filtron, Northborough, Mass.). The material retainedby the membrane was shown by radioimmunoassay and western blotting tocontain the L1 protein.

The retentate was applied to a high resolution affinity column (11.0 cmID×5.3 cm) of SP Spherodex (M)® resin (IBF, Villeneuve-la-Garenne,France) equilibrated in 20 mM sodium phosphate, pH 7.2, 0.5M NaCl.Following a wash with equilibration buffer and a step wash with 20 mMsodium phosphate, pH 7.2, 1.0M NaCl, the L1 protein was eluted with astep wash of 20 mM sodium phosphate, pH 7.2, 2.5M NaCl. Fractions werecollected during the washes and elution. Column fractions were assayedfor total protein by the Bradford method. Fractions were then analyzedby western blotting and SDS-PAGE with colloidal Coomassie detection.Fractions were also analyzed by radioinununoassay.

SP Spherodex fractions showing comparable purity and enrichment of L1protein were pooled.

Final product was analyzed by western blotting and SDS-PAGE withcolloidal Coomassie detection. The L1 protein was estimated to be >90%homogeneous. The identity of L1 protein was confirmed by westernblotting. The final product was filtered aseptically through a 0.22 μmmembrane and stored at 4° C. This process resulted in a total of 100 mgprotein.

Electron microscopy analysis is performed by Structure Probe (WestChester, Pa.). An aliquot of sample is placed on a 200 meshcarbon-coated copper grid. A drop of 2% phosphotungstic acid, pH 7.0 isplaced on the grid for 20 seconds. The grid is allowed to air dry priorto TEM examination. All microscopy is performed using a JEOL 100 CXtransmission electron microscope (JEOL USA, Inc.) at an acceleratingvoltage of 100 kV. The micrographs generated have a final magnificationof 100,000×.

Bradford Assay for Total Protein

Total protein was assayed using a commercially available Coomassie Plus®kit (Pierce, Rockford, Ill.). Samples were diluted to appropriate levelsin Milli-Q-H₂ O. Volumes required were 0.1 mL and 1.0 mL for thestandard and microassay protocols, respectively. For both protocols, BSA(Pierce, Rockford, Ill.) was used to generate the standard curve. Assaywas performed according to manufacturer's recommendations. Standardcurves were plotted using CricketGraph® software on a Macintosh IIcicomputer.

SDS-PAGE and Western Blot Assays

All gels, buffers, and electrophoretic apparatus were obtained fromNovex (San Diego, Calif.) and were run according to manufacturer'srecommendations. Briefly, samples were diluted to equal proteinconcentrations in Milli-Q-H₂ O and mixed 1:1 with sample incubationbuffer containing 200 mM DTT. Samples were incubated 15 min at 100° C.and loaded onto pre-cast 12% Tris-glycine gels. The samples wereelectrophoresed at 125 V for 1 hr 45 min. Gels were developed bycolloidal Coomassie staining using a commercially obtained kit(Integrated Separation Systems, Natick, Mass.).

For western blots, proteins were transferred to PVDF membranes at 25 Vfor 40 min. Membranes were washed with Milli-Q-H2O and air-dried.Primary antibody was polyclonal rabbit antiserum raised against aTrpE-HPV11 L1 fusion protein (gift of Dr. D. Brown). The antibodysolution was prepared by dilution of antiserum in blotting buffer (5%non-fat milk in 6.25 mM Na phosphate, pH 7.2, 150 mM NaCl, 0.02% NaN₃).Incubation was for at least 1 hour at 20-23° C. The blot was washed for1 min each in three changes of PBS (6.25 mM Na phosphate, pH 7.2, 150 mMNaCl). Secondary antibody solution was prepared by diluting goatanti-rabbit IgG alkaline phosphatase-linked conjugate antiserum (Pierce,Rockford, Ill.) in blotting buffer. Incubation proceeded under the sameconditions for at least 1 hour. Blots were washed as before and detectedusing a 1 step NBT/BCIP substrate (Pierce, Rockford, Ill.).

EXAMPLE 13

Preparation of Immunogenic Compositions

Purified VLP's are formulated according to known methods, such as by theadmixture of pharmaceutically acceptable carriers, stabilizers, or avaccine adjuvant. The immunogenic VLP's of the present invention may beprepared for vaccine use by combining with a physiologically acceptablecomposition such as, e.g. PBS, saline or distilled water. Theimmunogenic VLP's are administered in a dosage range of about 0.1 to 100mcg, preferably about 1 to about 20 mcg, in order to obtain the desiredimmunogenic effect. The amount of VLP per formulation may vary accordingto a variety of factors, including but not limited to the individual'scondition, weight, age and sex. Administration of the VLP formulationmay be by a variety of routes, including but not limited to oral,subcutaneous, topical, mucosal and intramuscular. Such VLP formulationsmay be comprised of a single type of VLP (i.e., VLP from HPV11) or amixture of VLP's (i.e, VLP's from HPV6, HPV11, HPV16 and HPV18).

An antimicrobial preservative, e.g. thimerosal, optionally may bepresent. The immunogenic antigens of the present invention may beemployed, if desired, in combination with vaccine stabilizers andvaccine adjuvants. Typical stabilizers are specific compounds, e.g.polyanions such as heparin, inositol hexasulfate, sulfatedbeta-cyclodextrin, less specific excipients, e.g. amino acids, sorbitol,mannitol, xylitol, glycerol, sucrose, dextrose, trehalose, andvariations in solution conditions, e.g. neutral pH, high ionic strength(ca. 0.5-2.0M salts), divalent cations (Ca²⁺, Mg²⁺). Examples ofadjuvants are Al(OH)₃ and Al(PO₄). The vaccine of the present inventionmay be stored under refrigeration or in lyophilized form.

EXAMPLE 14

Preparation of Antibodies to VLP

Purified VLP are used to generate antibodies. The term "antibody" asused herein includes both polyclonal and monoclonal antibodies as wellas fragments thereof, such as Fv, Fab and F(ab)2 fragments that arecapable of binding antigen or hapten. The antibodies are used in avariety of ways, including but not limited to the purification ofrecombinant VLP, the purification of native L1 or L2 proteins, and kits.Kits would comprise a compartmentalized carrier suitable to hold inclose confinement at least one container. The carrier would furthercomprise reagents such as the anti-VLP antibody or the VLP suitable fordetecting HPV or fragments of HPV or antibodies to HPV. The carrier mayalso contain means for detection such as labeled antigen or enzymesubstrates or the like. The antibodies or VLP or kits are useful for avariety of purposes, including but not limited to forensic analyses andepidemiological studies.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 38                                            - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 164 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 - GAAGATCTCA CAAAACAAAA TGTGGCGGCC TAGCGACAGC ACAGTATATG TG - #CCTCCTCC         60                                                                          - TAACCCTGTA TCCAAAGTTG TTGCCACGGA TGCTTATGTT AAACGCACCA AC - #ATATTTTA        120                                                                          #164               AGAC TTCTTGCAGT GGGTCATCCT TATT                            - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 156 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 - ATTCCATAAA AAAGGTTAAC AAAACTGTTG TGCCAAAGGT GTCAGGATAT CA - #ATACAGAG         60                                                                          - TATTTAAGGT GGTGTTACCA GATCCTAACA AATTTGCATT GCCTGACTCG TC - #TCTTTTTG        120                                                                          #      156         TTTG GTATGGGCAT GCATGT                                     - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 136 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 - ACATGCATGC ACAGGCCTAG AGGTGGGCCG GGGACAGCCA TTAGGTGTGG GT - #GTAAGTGG         60                                                                          - ACATCCTTTA CTAAATAAAT ATGATGATGT TGAAAATTCA GGGGGTTACG GT - #GGTAACCC        120                                                                          #   136                                                                       - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 127 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 - GTTAATGTAG GTATGGATTA TAAACAAACA CAATTATGCA TGGTTGGATG TG - #CCCCCCCT         60                                                                          - TTGGGCGAGC ATTGGGGTAA AGGTACACAG TGTAGTAATA CATCTGTACA GA - #ATGGTGAC        120                                                                          #         127                                                                 - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 125 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 - CCTTAGAACT TATTACCAGT GTTATACAGG ATGGCGATAT GGTTGACACA GG - #CTTTGGTG         60                                                                          - CTATGAATTT TGCTGATTTG CAGACCAATA AATCAGATGT TCCTCTTGAC AT - #ATGTGGCA        120                                                                          #           125                                                               - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 116 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                 - TGTAAATATC CAGATTATTT ACAAATGGCT GCAGACCCAT ATGGTGATAG AT - #TATTTTTT         60                                                                          - TATCTACGGA AGGAACAAAT GTTTGCCAGA CATTTTTTTA ACAGGGCTGG TA - #CCCC            116                                                                          - (2) INFORMATION FOR SEQ ID NO:7:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 124 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                 - GGGGTACCGT GGGGGAACCT GTGCCTGATG ATCTTTTAGT TAAGGGTGGT AA - #CAATCGCT         60                                                                          - CGTCTGTAGC GAGTAGTATA TATGTTCACA CCCCAAGCGG CTCTTTGGTG TC - #CTCTGAGG        120                                                                          #            124                                                              - (2) INFORMATION FOR SEQ ID NO:8:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 113 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                 - ATTGTTTAAT AAGCCATATT GGCTACAAAA AGCCCAGGGA CATAACAATG GT - #ATTTGTTG         60                                                                          - GGGTAATCAT CTGTTTGTTA CTGTGGTAGA TACCACACGC AGTACCAACA TG - #A               113                                                                          - (2) INFORMATION FOR SEQ ID NO:9:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 113 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                 - CATTATGTGC ATCCGTATCT AAATCTGCCA CATACACCAA TTCTGATTAT AA - #AGAGTACA         60                                                                          - TGCGTCATGT GGAAGAGTTT GATTTACAAT TTATTTTTCA ATTATGTAGC AT - #T               113                                                                          - (2) INFORMATION FOR SEQ ID NO:10:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 105 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                - ACATTGTCTG CTGAAGTAAT GGCCTATATT CACACAATGA ATCCCTCTGT TC - #TCGAGGAC         60                                                                          #                 105CC TCCCCCAAAT GGTACACTCG AGCGG                           - (2) INFORMATION FOR SEQ ID NO:11:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 155 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                - CCGCTCGAGG ATACCTATAG GTATGTGCAG TCACAGGCCA TTACCTGTCA AA - #AGCCCACT         60                                                                          - CCTGAAAAGG AAAAGCAAGA TCCCTATAAG GACATGAGTT TTTGGGAGGT TA - #ATTTAAAA        120                                                                          #      155         AATT GGATCAGTTT CCTTT                                      - (2) INFORMATION FOR SEQ ID NO:12:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 134 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                - GGGACGCAAG TTTTTGTTAC AAAGTGGATA TAGGGGACGG ACCTCTGCTC GT - #ACCGGTAT         60                                                                          - TAAGCGCCCT GCTGTTTCCA AACCCTCTAC TGCCCCTAAA CGTAAGCGCA CC - #AAAACTAA        120                                                                          #    134                                                                      - (2) INFORMATION FOR SEQ ID NO:13:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 154 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                - GAAGATCTTA CTTTTTAGTT TTGGTGCGCT TACGTTTAGG GGCAGTAGAG GG - #TTTGGAAA         60                                                                          - CAGCAGGGCG CTTAATACCG GTACGAGCAG AGGTCCGTCC CCTATATCCA CT - #TTGTAACA        120                                                                          #       154        AGGA AACTGATCCA ATTC                                       - (2) INFORMATION FOR SEQ ID NO:14:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 135 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                - ACTAGAAAAC TTTTCTTTTA AATTAACCTC CCAAAAACTC ATGTCCTTAT AG - #GGATCTTG         60                                                                          - CTTTTCCTTT TCAGGAGTGG GCTTTTGACA GGTAATGGCC TGTGACTGCA CA - #TACCTATA        120                                                                          #   135                                                                       - (2) INFORMATION FOR SEQ ID NO:15:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 125 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                - CCGCTCGAGT GTACCATTTG GGGGAGGCGA TAACCCAAAG TTCCAGTCCT CG - #AGAACAGA         60                                                                          - GGGATTCATT GTGTGAATAT AGGCCATTAC TTCAGCAGAC AATGTAATGC TA - #CATAATTG        120                                                                          #           125                                                               - (2) INFORMATION FOR SEQ ID NO:16:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 113 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                - TAAATTGTAA ATCAAACTCT TCCACATGAC GCATGTACTC TTTATAATCA GA - #ATTGGTGT         60                                                                          - ATGTGGCAGA TTTAGATACG GATGCACATA ATGTCATGTT GGTACTGCGT GT - #G               113                                                                          - (2) INFORMATION FOR SEQ ID NO:17:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 113 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                - GTATCTACCA CAGTAACAAA CAGATGATTA CCCCAACAAA TACCATTGTT AT - #GTCCCTGG         60                                                                          - GCTTTTTGTA GCCAATATGG CTTATTAAAC AATTGTGCCT CAGAGGACAC CA - #A               113                                                                          - (2) INFORMATION FOR SEQ ID NO:18:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 104 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                - AGAGCCGCTT GGGGTGTGAA CATATATACT ACTCGCTACA GACGAGCGAT TG - #TTACCACC         60                                                                          #104               TCAG GCACAGGTTC CCCCACGGTA CCCC                            - (2) INFORMATION FOR SEQ ID NO:19:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 136 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                - GGGGTACCAG CCCTGTTAAA AAAATGTCTG GCAAACATTT GTTCCTTCCG TA - #GATAAAAA         60                                                                          - AATAATCTAT CACCATATGG GTCTGCAGCC ATTTGTAAAT AATCTGGATA TT - #TACATACA        120                                                                          #   136                                                                       - (2) INFORMATION FOR SEQ ID NO:20:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 125 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                - GAGGAACATC TGATTTATTG GTCTGCAAAT CAGCAAAATT CATAGCACCA AA - #GCCTGTGT         60                                                                          - CAACCATATC GCCATCCTGT ATAACACTGG TAATAAGTTC TAAGGGCGGG CA - #GTCACCAT        120                                                                          #           125                                                               - (2) INFORMATION FOR SEQ ID NO:21:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 127 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                - ACAGATGTAT TACTACACTG TGTACCTTTA CCCCAATGCT CGCCCAAAGG GG - #GGGCACAT         60                                                                          - CCAACCATGC ATAATTGTGT TTGTTTATAA TCCATACCTA CATTAACCCT GT - #TATCCTGT        120                                                                          #         127                                                                 - (2) INFORMATION FOR SEQ ID NO:22:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 116 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                - TACCACCGTA ACCCCCTGAA TTTTCAACAT CATCATATTT ATTTAGTAAA GG - #ATGTCCAC         60                                                                          - TTACACCCAC ACCTAATGGC TGTCCCCGGC CCACCTCTAG GCCTGTGCAT GC - #ATGT            116                                                                          - (2) INFORMATION FOR SEQ ID NO:23:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 170 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                - ACATGCATGC CCATACCAAA CGTTGTGTTG TGGGATCAAA AAGAGACGAG TC - #AGGCAATG         60                                                                          - CAAATTTGTT AGGATCTGGT AACACCACCT TAAATACTCT GTATTGATAT CC - #TGACACCT        120                                                                          #             170TTGTTA ACCTTTTTTA TGGAATAATA AGGATGACCC                      - (2) INFORMATION FOR SEQ ID NO:24:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 150 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                - ACTGCAAGAA GTCTAGAACT GCTGGCATGA TAAAATATGT TGGTGCGTTT AA - #CATAAGCA         60                                                                          - TCCGTGGCAA CAACTTTGGA TACAGGGTTA GGAGGAGGCA CATATACTGT GC - #TGTCGCTA        120                                                                          #          150     TTTG TGAGATCTTC                                            - (2) INFORMATION FOR SEQ ID NO:25:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 27 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                #             27   CACA GGCCTAG                                               - (2) INFORMATION FOR SEQ ID NO:26:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 26 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                #              26  GCCC TGTTAA                                                - (2) INFORMATION FOR SEQ ID NO:27:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 45 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                #45                TGGG TTATCGCCTC CCCCAAATGG TACAC                           - (2) INFORMATION FOR SEQ ID NO:28:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 45 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                #45                GGGG AGGCGATAAC CCAAAGTTCC AGTCT                           - (2) INFORMATION FOR SEQ ID NO:29:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 45 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                #45                CAAA ATGTGGCGGC CTAGCGACAG CACAG                           - (2) INFORMATION FOR SEQ ID NO:30:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 35 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                #       35         TGGT TTTGGTACGT TTTCG                                      - (2) INFORMATION FOR SEQ ID NO:31:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 34 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                #        34        TAGT TTTGGCGCGC TTAC                                       - (2) INFORMATION FOR SEQ ID NO:32:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 31 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                #          31      CTTT CTCTTGTATC G                                          - (2) INFORMATION FOR SEQ ID NO:33:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 30 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                #           30     TGGT TCTCTTCCTC                                            - (2) INFORMATION FOR SEQ ID NO:34:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                #21                TGAA A                                                     - (2) INFORMATION FOR SEQ ID NO:35:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                #21                AGCG A                                                     - (2) INFORMATION FOR SEQ ID NO:36:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1521 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                - TTCCATGTGG CGGCCTAGCG ACAGCACAGT ATATGTGCCT CCTCCTAACC CT - #GTATCCAA         60                                                                          - AGTTGTTGCC ACGGATGCTT ATGTTAAACG CACCAACATA TTTTATCATG CC - #AGCAGTTC        120                                                                          - TAGACTTCTT GCAGTGGGTC ATCCTTATTA TTCCATAAAA AAGGTTAACA AA - #ACTGTTGT        180                                                                          - GCCAAAGGTG TCAGGATATC AATACAGAGT ATTTAAGGTG GTGTTACCAG AT - #CCTAACAA        240                                                                          - ATTTGCATTG CCTGACTCGT CTCTTTTTGA TCCCACAACA CAACGTTTGG TA - #TGGGCATG        300                                                                          - CACAGGCCTA GAGGTGGGCC GGGGACAGCC ATTAGGTGTG GGTGTAAGTG GA - #CATCCTTT        360                                                                          - ACTAAATAAA TATGATGATG TTGAAAATTC AGGGGGTTAC GGTGGTAACC CT - #GGACAGGA        420                                                                          - TAACAGGGTT AATGTAGGTA TGGATTATAA ACAAACACAA TTATGCATGG TT - #GGATGTGC        480                                                                          - CCCCCCTTTG GGCGAGCATT GGGGTAAAGG TACACAGTGT AGTAATACAT CT - #GTACAGAA        540                                                                          - TGGTGACTGC CCGCCCTTAG AACTTATTAC CAGTGTTATA CAGGATGGCG AT - #ATGGTTGA        600                                                                          - CACAGGCTTT GGTGCTATGA ATTTTGCTGA TTTGCAGACC AATAAATCAG AT - #GTTCCTCT        660                                                                          - TGACATATGT GGCACTGTAT GTAAATATCC AGATTATTTA CAAATGGCTG CA - #GACCCATA        720                                                                          - TGGTGATAGA TTATTTTTTT ATCTACGGAA GGAACAAATG TTTGCCAGAC AT - #TTTTTTAA        780                                                                          - CAGGGCTGGT ACCGTGGGGG AACCTGTGCC TGATGATCTT TTAGTTAAGG GT - #GGTAACAA        840                                                                          - TCGCTCGTCT GTAGCGAGTA GTATATATGT TCACACCCCA AGCGGCTCTT TG - #GTGTCCTC        900                                                                          - TGAGGCACAA TTGTTTAATA AGCCATATTG GCTACAAAAA GCCCAGGGAC AT - #AACAATGG        960                                                                          - TATTTGTTGG GGTAATCATC TGTTTGTTAC TGTGGTAGAT ACCACACGCA GT - #ACCAACAT       1020                                                                          - GACATTATGT GCATCCGTAT CTAAATCTGC CACATACACC AATTCTGATT AT - #AAAGAGTA       1080                                                                          - CATGCGTCAT GTGGAAGAGT TTGATTTACA ATTTATTTTT CAATTATGTA GC - #ATTACATT       1140                                                                          - GTCTGCTGAA GTAATGGCCT ATATTCACAC AATGAATCCC TCTGTTCTCG AA - #GACTGGAA       1200                                                                          - CTTTGGGTTA TCGCCTCCCC CAAATGGTAC ACTCGAGGAT ACCTATAGGT AT - #GTGCAGTC       1260                                                                          - ACAGGCCATT ACCTGTCAAA AGCCCACTCC TGAAAAGGAA AAGCAAGATC CC - #TATAAGGA       1320                                                                          - CATGAGTTTT TGGGAGGTTA ATTTAAAAGA AAAGTTTTCT AGTGAATTGG AT - #CAGTTTCC       1380                                                                          - TTTGGGACGC AAGTTTTTGT TACAAAGTGG ATATAGGGGA CGGACCTCTG CT - #CGTACCGG       1440                                                                          - TATTAAGCGC CCTGCTGTTT CCAAACCCTC TACTGCCCCT AAACGTAAGC GC - #ACCAAAAC       1500                                                                          #                1521AC A                                                     - (2) INFORMATION FOR SEQ ID NO:37:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1521 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                                - TTATGTGGCG GCCTAGCGAC AGCACAGTAT ATGTGCCTCC TCCTAACCCT GT - #ATCCAAAG         60                                                                          - TTGTTGCCAC GGATGCTTAT GTTACTCGCA CCAACATATT TTATCATGCC AG - #CAGTTCTA        120                                                                          - GACTTCTTGC AGTGGGTCAT CCTTATTTTT CCATAAAACG GGCTAACAAA AC - #TGTTGTGC        180                                                                          - CAAAGGTGTC AGGATATCAA TACAGAGTAT TTAAGGTGGT GTTACCAGAT CC - #TAACAAAT        240                                                                          - TTGCATTGCC TGACTCGTCT CTTTTTGATC CCACAACACA ACGTTTGGTA TG - #GGCATGCA        300                                                                          - CAGGCCTAGA GGTGGGCCGG GGACAGCCAT TAGGTGTGGG TGTAAGTGGA CA - #TCCTTTCC        360                                                                          - TAAATAAATA TGATGATGTT GAAAATTCAG GGAGTGGTGG TAACCCTGGA CA - #GGATAACA        420                                                                          - GGGTTAATGT AGGTATGGAT TATAAACAAA CACAATTATG CATGGTTGGA TG - #TGCCCCCC        480                                                                          - CTTTGGGCGA GCATTGGGGT AAAGGTAAAC AGTGTACTAA TACACCTGTA CA - #GGCTGGTG        540                                                                          - ACTGCCCGCC CTTAGAACTT ATTACCAGTG TTATACAGGA TGGCGATATG GT - #TGACACAG        600                                                                          - GCTTTGGTGC TATGAATTTT GCTGATTTGC AGACCAATAA ATCAGATGTT CC - #TATTGACA        660                                                                          - TATGTGGCAC TACATGTAAA TATCCAGATT ATTTACAAAT GGCTGCAGAC CC - #ATATGGTG        720                                                                          - ATAGATTATT TTTTTTTCTA CGGAAGGAAC AAATGTTTGC CAGACATTTT TT - #TAACAGGG        780                                                                          - CTGGCGAGGT GGGGGAACCT GTGCCTGATA CTCTTATAAT TAAGGGTAGT GG - #AAATCGCA        840                                                                          - CGTCTGTAGG GAGTAGTATA TATGTTAACA CCCCAAGCGG CTCTTTGGTG TC - #CTCTGAGG        900                                                                          - CACAATTGTT TAATAAGCCA TATTGGCTAC AAAAAGCCCA GGGACATAAC AA - #TGGTATTT        960                                                                          - GTTGGGGTAA TCAACTGTTT GTTACTGTGG TAGATACCAC ACGCAGTACC AA - #CATGACAT       1020                                                                          - TATGTGCATC CGTAACTACA TCTTCCACAT ACACCAATTC TGATTATAAA GA - #GTACATGC       1080                                                                          - GTCATGTGGA AGAGTATGAT TTACAATTTA TTTTTCAATT ATGTAGCATT AC - #ATTGTCTG       1140                                                                          - CTGAAGTAAT GGCCTATATT CACACAATGA ATCCCTCTGT TTTGGAAGAC TG - #GAACTTTG       1200                                                                          - GGTTATCGCC TCCCCCAAAT GGTACATTAG AAGATACCTA TAGGTATGTG CA - #GTCACAGG       1260                                                                          - CCATTACCTG TCAAAAGCCC ACTCCTGAAA AGGAAAAGCC AGATCCCTAT AA - #GAACCTTA       1320                                                                          - GTTTTTGGGA GGTTAATTTA AAAGAAAAGT TTTCTAGTGA ATTGGATCAG TA - #TCCTTTGG       1380                                                                          - GACGCAAGTT TTTGTTACAA AGTGGATATA GGGGACGGTC CTCTATTCGT AC - #CGGTGTTA       1440                                                                          - AGCGCCCTGC TGTTTCCAAA GCCTCTGCTG CCCCTAAACG TAAGCGCGCC AA - #AACTAAAA       1500                                                                          #                1521AC A                                                     - (2) INFORMATION FOR SEQ ID NO:38:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1519 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                                - TTCCATGTGG CGGCCTAGCG ACAGCACAGT ATATGTGCCT CCTCCCAACC CT - #GTATCCAA         60                                                                          - GGTTGTTGCC ACGGATGCGT ATGTTAAACG CACCAACATA TTTTATCATG CC - #AGCAGTTC        120                                                                          - TAGACTCCTT GCTGTGGGAC ATCCATATTA CTCTATCAAA AAAGTTAACA AA - #ACAGTTGT        180                                                                          - ACCAAAGGTG TCTGGATATC AATATAGAGT GTTTAAGGTA GTGTTGCCAG AT - #CCTAACAA        240                                                                          - GTTTGCATTA CCTGATTCAT CCCTGTTTGA CCCCACTACA CAGCGTTTAG TA - #TGGGCGTG        300                                                                          - CACAGGGTTG GAGGTAGGCA GGGGTCAACC TTTAGGCGTT GGTGTTAGTG GG - #CATCCATT        360                                                                          - GCTAAACAAA TATGATGATG TAGAAAATAG TGGTGGGTAT GGTGGTAATC CT - #GGTCAGGA        420                                                                          - TAATAGGGTT AATGTAGGTA TGGATTATAA ACAAACCCAG CTATGTATGG TG - #GGCTGTGC        480                                                                          - TCCACCGTTA GGTGAACATT GGGGTAAGGG TACACAATGT TCAAATACCT CT - #GTACAAAA        540                                                                          - TGGTGACTGC CCCCCGTTGG AACTTATTAC CAGTGTTATA CAGGATGGGG AC - #ATGGTTGA        600                                                                          - TACAGGCTTT GGTGCTATGA ATTTTGCAGA CTTACAAACC AATAAATCGG AT - #GTTCCCCT        660                                                                          - TGATATTTGT GGAACTGTCT GCAAATATCC TGATTATTTG CAAATGGCTG CA - #GACCCTTA        720                                                                          - TGGTGATAGG TTGTTTTTTT ATTTGCGAAA GGAACAAATG TTTGCTAGAC AC - #TTTTTTAA        780                                                                          - TAGGGCCGGT ACTGTGGGGG AACCTGTGCC TGATGACCTG TTGGTAAAAG GG - #GGTAATAA        840                                                                          - CAGATCATCT GTAGCTAGTA GTATTTATGT ACATACACCT AGTGGCTCAT TG - #GTGTCTTC        900                                                                          - AGAGGCTCAA TTATTTAATA AACCATATTG GCTTCAAAAG GCTCAGGGAC AT - #AACAATGG        960                                                                          - TATTTGCTGG GGAAACCACT TGTTTGTTAC TGTGGTAGAT ACCACACGCA GT - #ACAAATAT       1020                                                                          - GACACTATGT GCATCTGTGT CTAAATCTGC TACATACACT AATTCAGATT AT - #AAGGAATA       1080                                                                          - CATGCGCCAT GTGGAGGAGT TTGATTTACA GTTTATTTTT CAATTGTGTA GC - #ATTACATT       1140                                                                          - ATCTGCAGAA GTCATGGCCT ATATACACAC AATGAATCCT TCTGTTTTGG AG - #GACTGGAA       1200                                                                          - CTTTGGTTTA TCGCCTCCAC CAAATGGTAC ACTGGAGGAT ACTTATAGAT AT - #GTACAGTC       1260                                                                          - ACAGGCCATT ACCTGTCAGA AACCCACACC TGAAAAAGAA AAACAGGATC CC - #TATAAGGA       1320                                                                          - TATGAGTTTT TGGGAGGTTA ACTTAAAAGA AAAGTTTTCA AGTGAATTAG AT - #CAGTTTCC       1380                                                                          - CCTTGGACGT AAGTTTTTAT TGCAAAGTGG ATATCGAGGA CGGACGTCTG CT - #CGTACAGG       1440                                                                          - TATAAAGCGC CCAGCTGTGT CTAAGCCCTC TACAGCCCCC AAACGAAAAC GT - #ACCAAAAC       1500                                                                          #                 151 - #9                                                    __________________________________________________________________________

What is claimed is:
 1. An isolated and purified DNA molecule encodinghuman papillomavirus type 11 L1 protein, the DNA molecule being free ofinternal transcription termination signals which are recognized byyeast.
 2. The DNA molecule of claim 1 having the sequence shown in FIG.8 SEQ. ID. NO.38.
 3. An expression vector comprising the DNA molecule ofclaim
 1. 4. A composition comprising the DNA molecule of claim 1, or RNAcomplementary to the DNA molecule of claim
 1. 5. An expression vectorcomprising the DNA molecule of claim
 2. 6. The expression vector ofclaim 5 which is D362-1.
 7. A nucleic acid encoding human papillomavirustype 11 L1 protein, the nucleic acid molecule being free from internaltranscription termination signals which are recognized by yeast.